The present invention relates to a method for rapid and easy quantitative measurement of a substrate contained in a sample such as blood, urine and fruit juice with high accuracy.
A conventional simple method for quantitating a specific component in a sample solution with no dilution or agitation of the sample solution is to cause the specific component to react with an oxidoreductase whose substrate corresponds to the specific component in the presence of an electron mediator or electron acceptor, followed by electrochemical oxidation of the electron mediator which has been reduced by this enzyme reaction, thereby to determine the oxidation current flowing during this electrochemical oxidation.
This method normally uses a biosensor as disclosed in the Japanese Laid-Open Patent Publication Hei 3-202764.
The biosensor is produced by first forming an electrode system having a working electrode and a counter electrode on an electrically insulating base plate by a screen printing method or the like, subsequently forming a reaction layer including an oxidoreductase and an electron mediator above the electrode system, and finally bonding a cover and a spacer to the electrically insulating base plate.
With this biosensor, various specific components can be quantitated by varying the oxidoreductase.
Here, a glucose sensor will be described as an example of biosensor.
Conventionally known method for quantitative measurement of glucose is a system comprising a combination of glucose oxidase with an oxygen electrode or a hydrogen peroxide electrode (e.g., "Biosensor", ed. by Shuichi Suzuki, Kodansha, Japan).
Glucose oxidase selectively oxidizes a substrate .beta.-D-glucose to D-glucono-.delta.-lactone by utilizing oxygen dissolved in a sample solution as an electron mediator. When the substrate is oxidized by the glucose oxidase, the oxygen used as the electron mediator is reduced to hydrogen peroxide. The glucose concentration can be quantitated either by measurement of the volume of oxygen consumed during this reaction using an oxygen electrode or by measurement of the volume of hydrogen peroxide produced using a hydrogen peroxide electrode of platinum or the like.
However, this method has a drawback that the measurement is largely affected by the concentration of oxygen contained in a sample solution, depending on the measuring object. This system has another drawback that the system cannot function in the absence of oxygen.
To overcome these problems, a type of glucose sensor has been developed which includes an organic compound or a metal complex such as potassium ferricyanide, ferrocene derivatives, quinone derivatives, etc. as electron mediator, in place of oxygen.
This biosensor can carry a known amount of glucose oxidase on an electrode system, together with an electron mediator in their stabilized state. As a result, the electrode system can be integrated with the reaction layer almost in dry state.
Such biosensor is normally disposable and facilitates measurement of the concentration of glucose by a simple instillation of a measuring sample at a sensor chip mounted in a measurement device. Therefore, this biosensor has been attracting much attention recently.
As described above, the substrate in a sample can be quantitated based on the current flowing across the electrodes during oxidation of the electron mediator which has been reduced by a series of enzyme reaction.
If the oxidation current value is measured with a two-electrode system comprising a working electrode and a counter electrode, then the presence of an electron mediator in oxidized state which must be reduced on the counter electrode becomes mandatory.
When the measuring sample is predicted to have a low concentration of substrate, it becomes unnecessary to secure the presence of such electron mediator in oxidized state, because the amount of oxidized electron mediator to be reduced by enzyme reaction is small.
However, when the measuring sample is predicted to have a high concentration of substrate, most of the electron mediator in oxidized state is reduced by enzyme reaction, resulting in a deficiency of oxidized electron mediator which can be reduced on the counter electrode. This renders the reduction on the counter electrode to show a rate-determining step, affecting the resultant current value.
Moreover, depending on sample, an easy-to-oxidize substance may be present that is oxidized to induce an oxidation current at the same time when the electron mediator in reduced state is oxidized on the electrode, producing a positive error in the current value measured. Furthermore, a high concentration of substrate may vary the oxidation current value.